Combining multiplex gene editing and doubled haploid technology in maize

Summary A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, i.e. the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction, and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

) are shown.(SAM) and young stem from the Maize Gene Expression Atlas (Stelpflug et al., 2016) are plotted as a heat map.The heatmap and the hierarchical clustering of the genes presented here was created using the clustermap function of the seaborn package (Hunter, 2007;Waskom, 2021).DAS, days after sowing; Vn, vegetative stage corresponding to the number of emerged leaves; VT, vegetative tasseling; DAP, days after pollination; R2, reproductive 2 stage.All samples are from field-grown plants, except coleoptile (GH, greenhouse).or with each other (F1; in the BREEDIT strategy called "interscript cross"), heterozygous progeny is used for haploid induction and the generation of doubled haploids via embryo rescue doubling (ERD).Self-crossing those doubled haploids results in a collection of immortal DH lines, differing in edit combinations as identified by HiPlex genotyping.(c) A first possible goal of GEDH is to identify a minimal causal genotype based on replicated phenotyping of various DH lines (Purify for gene deconvolution).If the number of target genes (g) is high, the number of DH lines feasible to generate is far smaller than all possible combinations (n < 2 g ).Then, a phenotypic screen only partially covers the mutation combination space, but may still identify a DH line with reduced number of mutations and with the desired phenotype.This stage can be followed up by a complete screen after iteratively narrowing down the number of targets (g).When the number of target genes (g) is relatively small, a collection of n DH lines can cover all possible combinations in a complete phenotypic screen (n >> 2 g ).(d) A second possible goal is to generate and identify in a targeted way a specific, predefined homozygous genotype.For instance, a set of genes for which all homozygous gene edits are desired may be defined based on i) association data; ii) all genes in a QTL region; or iii) a gene family.The minimal population size (n min , i.e. collection of GEDH lines) required to identify a favorable genotype with a given probability using a DH strategy can be estimated (Lübberstedt & Frei, 2012).Here, we calculated the relationship between the probability to identify a particular genotype for increasing number of targets (g), given a feasible population size in four scenarios (146 or 1000 DH lines, or 1000 or 9430 plants obtained after a self-cross).

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Fig. S2 Maize (Zea mays) cob and isolated embryos after a cross of B104 (female) and RWS-GFP (male).(a) Cob harvested 14 days after an in vivo haploid induction cross.At this stage, embryos were isolated and haploids separated from diploids.(b) Haploid maize embryos (indicated by white arrowheads) were scored by the absence of GFP expression (RWS-GFP genome elimination).Diploids show GFP expression with the RWS-GFP genome still present.

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Fig. S3 Examples of three different flow cytometry outcomes after Zea mays (maize) haploid doubling.Graphs represent DNA content measured by CyFlow®ML cytometer (Partec), x-axes represent DNA content (fluorescent channel 1, FL1), y-axis shows event count.Example histogram of a (a) haploid plant, (b) diploid plant, and (c) mixoploid plant.Peaks are labeled with their respective ploidy number.Below the graphs, ploidy scores and the plant numbers (as seen in TableS3) are shown.

Fig. S4
Fig. S4 Phenotypic analysis of Zea mays (maize) SCRIPT 4 DH lines.Genotypes and corresponding phenotypes observed in DH SCRIPT 4 lines homozygous for various combinations of out-of-frame alleles (green squares) and in-frame mutated alleles (gray squares); the size of the indel (in bp) is indicated in the squares.White squares indicate that the wild-type reference (REF) allele was identified by genotyping.Each row represents an independent DH line.Boxplots with jittered data points on the right display measurements of (a) final leaf 3 length (FLL3) and (b) final leaf width (FLW3) for edited plants compared with non-edited control plants (wild-type B104 and two wild-type doubled haploids (HIC01 and HIC03)).DH lines are sorted from lowest to highest mean FLL3 (a) or FLW3 (b).24 to 29 seeds were sown for each DH line; n, number of germinated plants phenotyped.The compact letter display shows the result of the pairwise comparisons of the Wilcoxon rank sum test (significance level of 5% with Holm correction).

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Fig. S5 Phenotypic screen of PLA3 in Zea mays (maize) inter-script DH lines.Genotypes and corresponding phenotypes observed in DH1 inter-script plants homozygous for various combinations of out-of-frame alleles (green squares) and in-frame mutated alleles (gray squares).White squares indicate that the wild-type reference (REF) allele was identified by genotyping.Each row represents an independent DH line.Boxplots with jittered data points on the right display measurements of pseudo leaf 3 area (PLA3) for edited plants compared with non-edited control plants (wild-type B104, EDITOR 1 without SCRIPT (ED1) and a wild-type doubled haploid (HIC01)).DH lines are sorted from lowest to highest mean PLA3.Eight to twelve DH1 seeds were sown for each DH line and 30 seeds for three control lines; n, number of germinated plants phenotyped.The compact letter display shows the result of the pairwise comparisons of the Wilcoxon rank sum test (significance level of 5% with Holm correction).Absent squares for TCP42 indicate missing data due to low-quality amplicons.The FLL3 and FLW3 components of PLA3 are plotted individually in Fig. S6.

Fig. S7
Fig. S7 Power analysis for the use of PLA3 in replicated phenotyping of Zea mays (maize).X-axisshows the number of plants (n) required to show a 2%, 5%, or 10% significant difference in PLA3 with a power of 80% (dashed horizontal line).Based on B104 wild-type variation seen in PLA3 measurements over the phenotypic experiments in this manuscript, assumption of normality and using a t-test.

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Fig. S8 Fig. S8.Phenotypic analysis of Zea mays (maize) DH1 and DH2 plants.Genotypes and corresponding phenotypes observed in DH1 and DH2 generations for four different homozygous edited lines and non-edited control lines (HIC01).Out-of-frame mutated alleles (green squares), in-frame mutated alleles (grey squares) and reference alleles (white squares), the size of the indel (in bp) is indicated in the squares.On the left, each row represents the genotype of a line (DH1 or DH2), on the right, corresponding boxplots with jittered data points display measurements of (a) final leaf 3 length (FLL3) and (b) final leaf width (FLW3).24-35 seeds of each line were sown; n, number of germinated plants phenotyped.The compact letter display shows the result of the pairwise comparisons of the Wilcoxon rank sum test (significance level of 5% with Holm correction).

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Fig. S9 Expression of Zea mays (maize) CKX genes targeted using SCRIPT 2. For each CKX gene, log10 transformed FPKM values for internode and the organ groups leaf, shoot apical meristem

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Fig. S10 Overview of possible GEDH strategies and goals.(a) Populations of mutants are made covering the target gene (g) space either with one SCRIPT with many targets (Overshoot) or